1. Field of the Invention
The present approach relates to methods for producing fats and oils. Specifically, the present approach pertains to prolonging the enzymatic activity of an enzyme used for transesterification or esterification of a substrate for the production of fats and oils by purification of the substrate prior to transesterification or esterification.
2. Related Art
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are able to catalyze a variety of reactions. Such enzymes are commercially available from a broad range of manufacturers and organisms, and are useful in catalyzing reactions with commodity oils and fats. See, e.g., Xu, X., “Modification of oils and fats by lipase-catalyzed interesterification: Aspects of process engineering,” in Enzymes in Lipid Modification, 190-215 (Bornscheuer, U. T., ed., Wiley-VCH Verlag GmbH, Weinheim, Germany, 2000). Lipases are useful to hydrolyze glycerides such as triacylglycerols and phosphatides. They are also useful in the synthesis of esters from industrial fatty acids and alcohols. In addition, lipases are useful for alcoholysis (exchanging alcohols bound to esters) for products such as biodiesel and partial glycerides. Lipases can also be used to catalyze acyl-exchange reactions such as interesterification (also known as transesterification) of mixed ester substrates to create unique blends of triacylglycerols with desired functional characteristics.
Biocatalysts such as lipases are also attractive due to their use under mild operating conditions and their high degrees of selectivity. Biocatalysts also offer synthetic routes which avoid the need for environmentally harmful chemicals.
Lipases are further useful for the manufacture of specialty glycerides. For example, 1,3-specific lipases are useful in the manufacture of 1,3-diglycerides, as described, for example, in U.S. Pat. No. 6,004,611.
The transesterification reaction has also become an important solution to a recently identified threat to human health: trans fatty acids. These trans fatty acids were long desired for their functional characteristics in food use and have been produced on commodity scale by partial hydrogenation of vegetable oils. Thus, they have been readily available and relatively inexpensive for decades. Currently, suppliers of food products are seeking fats to replace partially hydrogenated vegetable oil, preferably at comparable prices or lower. Transesterification of properly selected fats and oils can provide fats to replace partially hydrogenated vegetable oil. If such fats are produced by transesterification of fats and oils free from trans fatty acids, trans fatty acids will be substantially absent from the transesterified fat. Proper selection of fatty acid compositions of starting fats and oils will provide proper functionality in the transesterified replacement fats for partially hydrogenated oil advantageously synthesized by lipase-catalyzed interesterification.
The stability of biocatalysts such as lipases is most conveniently expressed in terms of half-life, which is the time after which the initial catalyst activity has decreased to half the original value. Diks, Rob M. M., “Lipase stability in oil,” Lipid Technology, 14(1): 10-14 (2002). Another way to express enzyme stability is the productivity of the enzyme, which is measured by the amount of the product per unit enzyme (g oil produced/g enzyme), during the first half-life. Typical lipase half-lives in interesterification reactions are seven days. See, e.g., Huang, Fang-Cheng and Ju, Yi-Hsu, “Interesterification of palm midfraction and stearic acid with Rhizopus arrhizus lipase immobilized on polypropylene,” Journal of the Chinese Institute of Chemical Engineers, 28(2): 73-78 (1997); Van der Padt, A. et al., “Synthesis of triacylglycerols. The crucial role of water activity control,” Progress in Biotechnology, 8 (Biocatalysis in Non-Conventional Media): 557-62 (1992). Half-lives vary greatly depending on the lipases themselves.
However, half-lives also vary depending on the quality of the substrates. When biocatalysts such as enzymes are used, components in the substrate mixture may diminish the effective lifetime of the catalyst. In continuous operations, the ratio of substrate processed to enzyme is very large, so minor components of oil can have a cumulative deleterious effect on enzyme activity. Several oxidation compounds in oil, such as hydroperoxides and secondary oxidation products (e.g., aldehydes or ketones), may cause significant lipase inactivation in oils. See, e.g., Pirozzi, Domenico, “Improvement of lipase stability in the presence of commercial triglycerides,” European Journal of Lipid Science and Technology 105(10): 608-613 (2003); Gray, J. I., “Measurement of Lipid Oxidation: A Review,” J. Amer. Oil Chem. Soc. 55: 539-546 (1978); U.S. Patent Application Publication No. 2005/0014237 A1, and publications cited therein. Oxidation products include oxidative species that initiate self-propagated radical reaction pathways, or other reactive oxygen species (such as peroxides, ozone, superoxide, etc.). These and other constituents which cause or arise from fat or oil degradation can result in enzyme degradation. The presence of water and other substances can also strongly influence the activity of lipases used in transesterification. See, e.g., Jung, H. J. and Bauer, W., “Determination of process parameters and modeling of lipase-catalyzed transesterification in a fixed bed reactor,” Chemical Engineering & Technology, 15(5): 341-8 (1992). Some metal ions (Mg2+ and Fe2+) have also been cited as inhibitors for some lipases. However, the processes and causative factors by which lipases become inactive are not completely understood.
It has been observed that using different batches of the same feedstock in a lipase-catalyzed oil gave wide variations in lipase half-life. Diks, Rob M. M., “Lipase stability in oil,” Lipid Technology, 14(1): 10-14 (2002). No relationship was found between lipase half-life and the oil's PV or the para-anisidine value (PAV). In addition, no correlation between metal levels (Fe and Cu), polymerized glycerides, or phospholipids and lipase half-life could be established.
An investigation into the cause of loss of activity of immobilized lipase in the acidolysis of high oleic sunflower oil with stearic acid determined that oxidation products increased the rate of deactivation, but removal of oxidation products from the oils prevented activity loss. Nezu, T. et al., “The effect of lipids oxidation on the activity of interesterification of triglyceride by immobilized lipase,” in Dev. Food Eng., 6th Proc. Int. Congr. Eng. Food, 591-3 (Yano, T. et al., eds., Blackie, Glasgow, 1994). Immobilized lipases incubated with 2-unsaturated aldehydes (typically formed as secondary oxidation products in the oxidative breakdown of oils) lost their catalytic activity. Linoleic acid hydroperoxides at levels of PV>5 meq/kg causes loss of lipase activity, and the rate of enzyme inactivation increases as PV increased; the mechanism of enzyme inactivation was the generation of free radicals in the enzyme as the peroxides decomposed. Wang, Y. and Gordon, M. H., “Effect of lipid oxidation products on the transesterification activity of an immobilized lipase,” Journal of Agricultural and Food Chemistry, 39(9): 1693-5 (1991). When oxidized lipids were separated from a sample of palm oil and fractionated, it was demonstrated that fractions exhibiting high degrees of inactivation could be isolated, but the inhibitory compounds were not identified. Id.
Rapid lipase activity decrease during continuous lipase catalyzed reactions is common. See, e.g., Ferreira-Dias, S. et al. “Recovery of the activity of an immobilized lipase after its use in fat transesterification,” Progress in Biotechnology, 15 (Stability and Stabilization of Biocatalysis): 435-440 (1998); Diks, Rob M. M., “Lipase stability in oil,” Lipid Technology, 14(1):10-14 (2002).
Several methods have been tried to eliminate loss of activity or to recover activity from inactivated lipase.
a) Recovery of lipase activity lost in transesterification reactions was carried out by washing the lipase preparation with hexane and adjusting the water activity of the preparation to 0.22. Ferreira-Dias, S. et al. “Recovery of the activity of an immobilized lipase after its use in fat transesterification,” Progress in Biotechnology, 15 (Stability and Stabilization of Biocatalysis): 435-440 (1998). Although the mechanism was unknown, this type of activity recovery is consistent with activity loss caused by accumulation of inhibitory compounds such as lipid oxidation products. Id.
b) Reducing the water activity of a transesterification substrate (crude palm oil/degummed rapeseed oil) from 280 ppm to 60 ppm was accompanied by an increase of immobilized lipase half-life from 10 hours to 100 hours. Huang, Fang-Cheng and Ju, Yi-Hsu, “Interesterification of palm midfraction and stearic acid with Rhizopus arrhizus lipase immobilized on polypropylene,” Journal of the Chinese Institute of Chemical Engineers, 28(2):73-78 (1997).
c) Lipase half life has been increased by immobilizing certain compositions with lipase. For example, the half life of lipase immobilized on controlled pore silica increased fivefold when PEG-1500 was co-immobilized with the lipase. Soares, C. M. F. et al., “Selection of stabilizing additive for lipase immobilization on controlled pore silica by factorial design,” Applied Biochemistry and Biotechnology, 91-93(Symposium on Biotechnology for Fuels and Chemicals, 2000):703-718 (2001).
d) JP 11-103884 described the addition of small amounts (0.01-5 wt %) of phospholipids to an immobilized Alcaligenes lipase caused a ten-fold increase in lipase half life.
e) Others have prolonged lipase half-life via pre-treatment of the substrate oil. JP 08-140689 A2 describes the use of Duolite A-7 ion exchange resin to treat a blend of palm oil with ethyl stearate prior to interesterification using and immobilized Rhizopus lipase to increase the half life from 3 days to 8 days. Duolite A-7 is an anion exchange resin containing amino groups. JP 08-140689 A2 also describes pre-treatment of substrate oils with proteins or peptides containing a large number of basic amino acid residues such as histone, protamine, lysozyme or polylysine. JP 08-140689 A2 states that amino groups are believed to react with aldehydes or ketones (secondary oxidation products) to form a Schiff base; and that such secondary oxidation products are believed to be a factor in lipase inactivation.
f) JP 02-203789 A2 describes extending the half life of immobilized lipase by pre-treatment of the substrate with an alkaline substance. When an equal mixture of rapeseed oil and palm olein was interesterified on a column of lipase immobilized on Celite 535, the half life of the lipase was 18 hours. When the substrate was mixed with a solution of potassium hydroxide (5 mL/kg substrate) the half life of the enzyme activity was 96 h. An alternative approach is to treat celite with sodium hydroxide and mix this into the same substrate mixture. Using this approach, lipase half life was extended to 33 hours. JP 02 203790 A2.
g) It has been demonstrated that, Novozyme 435 is more affected by secondary oxidation products than by hydroperoxides (Pirozzi, Domenico, “Improvement of lipase stability in the presence of commercial triglycerides,” European Journal of Lipid Science and Technology 105(10):608-613 (2003)). With this lipase, it has been shown that lipase sulphydryl groups interact with two secondary oxidation product aldehydes, 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). By neutralizing 4-HNE and MDA in oil with albumin, enzyme stability was increased.
h) U.S. Patent Application No. 2003/0054509 describes the use of unmodified purification media (e.g., silica gel) to increase enzymatic half-life. U.S. Patent Application No. 2005/0014237 describes the use of deodorization processes to increase enzymatic half-life.
Hence, there is a long-felt need in the art of enzymatic catalysis for solutions to this activity loss. See also Diks, Rob M. M., “Lipase stability in oil,” Lipid Technology, 14(1):10-14 (2002); Wang, Y. and Gordon, M. H., “Effect of lipid oxidation products on the transesterification activity of an immobilized lipase,” Journal of Agricultural and Food Chemistry, 39(9):1693-5 (1991). The time period over which lipase retains its enzymatic activity is an important cost consideration in lipase-catalyzed interesterification. The loss of effective enzyme activity is detrimental to industrial processing due to the cost of replacement enzyme and production time needed to change enzymes, switch columns, and stabilize a new column. Thus, the extension of enzyme half-life is extremely critical for the successful commercialization of enzymatic interesterification. This long-felt need is a primary barrier to the expansion of enzyme catalyzed reactions for production of commodity or “bulk” chemicals.
Although most of the mechanisms of lipase inactivation and its prevention are poorly understood at present, the present approach describes an effective solution to preventing lipase degradation and increasing its productivity and half-life.